Efficient intracellular delivery of proteins by a multifunctional chimaeric peptide in vitro and in vivo

Protein delivery with cell-penetrating peptide is opening up the possibility of using targets inside cells for therapeutic or biological applications; however, cell-penetrating peptide-mediated protein delivery commonly suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting in vivo efficacy. Here, we present an intracellular protein delivery system consisting of four modules in series: cell-penetrating peptide, pH-dependent membrane active peptide, endosome-specific protease sites and a leucine zipper. This system exhibits enhanced delivery efficiency and serum tolerance, depending on proteolytic cleavage-facilitated endosomal escape and leucine zipper-based dimerisation. Intravenous injection of protein phosphatase 1B fused with this system successfully suppresses the tumour necrosis factor-α-induced systemic inflammatory response and acetaminophen-induced acute liver failure in a mouse model. We believe that the strategy of using multifunctional chimaeric peptides is valuable for the development of cell-penetrating peptide-based protein delivery systems, and facilitate the development of biological macromolecular drugs for use against intracellular targets.

1) The most significant issue of the paper is shown in Supplemental Figure 1. These data show very clearly that better delivery does not mean that individual cells get more protein delivered to them. All constructs tested, including the very poor ones, deliver a similar amount of cargo to individual cells. The better constructs simply deliver this amount of cargo to more cells. This fact is not addressed in the main paper, yet it holds important clues to something very fundamental about protein delivery that we do not understand. Many researchers have observed the same phenomenon. Delivery is stochastic, and cooperative at the cell level, not at the individual endosome level. Improving protein delivery to cells is not about improving the efficiency of release from individual endosomes. It is not about delivery of more protein to individual cells. With respect to cargo delivery, cells act cooperatively. Improving delivery is about delivering a bolus of protein to a greater fraction of cells. Yet, this entire paper (and most other papers on cargo delivery) presents the problem and discusses the results it as if the modifications carried out incrementally improve the amount of protein delivered to each cell at the level of individual endosomes. This is not correct. The fundamentally important observation in Supplemental Fig 1 must be a central part of the whole paper. Introduction, results, and discussion.
2) The fluorescence microscopy images shown for delivery of full length GFP seem not to show the same effect as described in point 1 above. Is this true? The authors should explain this.
3) One serious problem with almost all protein delivery assays is a lack of absolute measures of delivery. For example, Figures 1c and 1d show mean fluorescence intensity as a ratio of intensity observed with various constructs, to that observed with a contract that delivers little to no GFP1-10. Relative MFI is the ratio of a measurement over a small number. The reader of this paper needs to know what MFI=10 means in terms of absolute amounts. One could lyse cells, and use absolute fluorescence of the lysate compared to standard solutions of known concentrations of GFP to determine the absolute amount of GFP1-10 delivered. But given the stochastic nature of delivery (Supplemental Figure 1) a microscopic technique might be better suited. This problem of quantitation is even worse for enzymes because their effect is greatly amplified.
4) The writing syntax and sentence structure is very poor throughout the manuscript. The revised paper will need significant editing and polishing of the English. To provide one example, the opening paragraph of the discussion has several sentences that are nonsensical at present.
4) The paper contains a number of statements that are essentially unsupported speculation. I feel that they should be removed if they cannot be supported by some evidence from the literature or from these experiments. Such speculations include: Line 119-120 "The CPP dimer might engage with the cellular membrane through multivalent interactions, but with, serum albumin in a monovalent manner; thus, the competitiveness of the serum is alleviated." Lines 253-256 "Unexpectedly, endosomal escape efficiency of this dimer is quite low than observed at co-incubation approach of dfTAT30. The reason for this is unclear, it is possible that the effect of cargo molecules on the interaction between TAT with BMP (bis(monoacylglycero)phosphate)." 5) The animal experiments are very impressive and add a lot to this work. Again, we lack any quantitative measure of delivery. Is there a way to determine how much enzyme must be delivered to prevent the acute damage that drives the observed phenomenon? 6) Can the authors use the Cy5 fluorescence to determine the amount of enzyme delivered to cells? Can fluorescence microscopy be done on tissue sections to directly demonstrate and quantify cargo delivery to cells in vivo.
Reviewer #2: Remarks to the Author: Yu et al. report on a novel CPP(TAT)-based intracellular protein delivery system (eTAT) consisting of a CPP, PMAP, endosome-specific protease sites and a leucine zipper, showing enhanced cytosolic protein, phosphatase 1B (Ppm1b), delivery efficiency and serum tolerance in vitro and in vivo without toxicity, suppressing the TNF-α-induced systemic inflammatory response and acetaminophen-induced acute liver failure in the mouse model.
They provide a strategy to facilitate the development of CPP-based delivery platform on proteins for intracellular targets. The work is well performed and timely.
However, my concern is that it is difficult to see clear advantage of this method as compared to previously published protein delivery systems, e.g. TAT peptide and multiple others. I suggest to carry out additional studies in order to confirm such advantage.
Reviewer #3: Remarks to the Author: Authors have designed and refined Tat-peptide-based vector for delivering into cells protein cargoes covalently coupled to peptide. First, the widely used CPP was supplemented with PMAP sequence, and two protease cleaving sites for facilitating release of cargo proteins from endosomal entrapment. Later the dimerisation-inducing motif was included in peptide vector to increase the uptake by cells and reduce sensitivity towards serum albumin that is known to inhibit Tatmediated delivery into cells. The efficiency of elaborated eTAT vector was verified by delivering GFP and its split version into model cell-lines. Finally, eTAT was used for delivery of Pmp1b to suppress TNF-induced necroptosis in vitro and in vivo. Moreover, eTAT-Pmp1b also increased survival of mice in acetaminopheninduced acute liver failure model.
Major comments: 1. Authors claim that the used strategies for improving Tat-peptide mediated delivery are novel. However, higher delivery efficacy of multivalent CPPs compared to monovalent peptide has been earlier demonstrated by several laboratories e.g. of Futaki and Gariepy. Their works have to be referred to in introduction. The idea of using cleavage by protease for activation of CPP construct has been repeatedly exploited in the field of CPPs, and was pioneered by R. Tsien group, who perhaps also deserves mentioning. 2. Fig. 1a, a scheme of recombinant cell-penetrating protein, does not reveal where the polyhistidine tag that is used for purification of protein, is placed in the sequence. Please add! All data in manuscript suggest that histidine tag is still present on all used protein constructs, and it is not removed. Histidine residues are known to serve as a proton sponge and facilitate escape from endosomes, was its impact considered? 3. Fig 1h. The intensity of GFP signal is substantially higher for TI-GFP compared to T-GFP after incubation MA104 cells with the respective protein constructs. The efficiency of cationic CPPs is dependent on the charge of peptide (R and K in sequence). Addition of INF7 to TAT sequence reduces the charge of targeting sequence from 8 to 4, which typically strongly impairs internalisation of peptide/protein construct. Here the fluorescence microscopy shows oppositeprotein carrying the transducing sequence with lower positive charge is taken up by cells more efficiently than one with higher charge. Please explain this discrepancy in discussion part. Please show more than 1 or 2 cells in new version of Fig 1h. 4. Line 123 … As analyzed by SDS-PAGE, T-GFP and TL-GFP are monomers and dimers respectively (Fig. 2b). It is highly improbable that in SDS-PAGE the latter protein construct is in the form of dimer only. The Supplementary Figures 5, 6 and 8 show quite opposite. 5. Line 191 … Twenty-four hours after administration, eTAT-Ppm1b was mostly distributed in the caecum … This is rather surprising observation. Has that been shown for other CPPs or CPP-protein constructs earlier? Please comment on this in discussion. 6. Line 234 … the endocytosis of the cargo was mediated by the interaction between TAT and the cell membrane (Fig. 5a) Does TAT peptide/motif interact directly with membrane? It has been repeatedly shown that proteins are involved in interaction and penetration of this CPP across membranes 7. Discussion contains multiple factual errors and difficult-to-understand sequences. It requires revision with focussing on obtained results and leaving out ample putative developments of presented protein delivery vector.
Specific comments: 1. Line 52-53 … After endocytosis mediated by CPP, the majority (approximately 90%) of the cargos are entrapped inside endosomes. Does it mean that 10% liberates and is active? Please provide reference for 90%. In general, one or two logs lower escape/activity is considered realistic. 2. Line 70-72 … Therefore, we believe the eTAT system or its improved version composed of multifunctional chimeric peptide has potential to become a major enabling strategy for protein delivery in biological research and therapeutic application. Exaggeration, considering how large quantities of protein construct (per kg) were required to achieve effect in mouse model. 3. In Figure 1e type of actin? The level of actin varies substantially between lines. Please specify (in Materials and Methods) how the amount of protein that was applied to gel was equalised. 4. Figure 1f. Fluorescence of GFP in split assay peaks at 9 h time-point and starts to decrease from there, which occurs surprisingly quickly. Please comment this in discussion. 5. Lines 118-119 … homodimerization capacity was employed to mediate the dimerization of the CPP-fused proteins. This approach has been used with CPPs earlier. Please add reference. 6. Lines 119-121 … The CPP dimer might engage with the cellular membrane through multivalent interactions, but with, serum albumin in a monovalent manner; thus, the competitiveness of the serum is alleviated. What gives ground for this assumption? Has it been shown experimentally? For verifying this assumption, the authors are advised to analyse the elution of dimeric protein construct (after incubation with serum or not) from SEC column. Comparison of that with elution profiles of monomeric from should reveal, whether both proteins associate only one molecule of albumin. 7. Line 170 … eTAT showed the highest efficiency for Ppm1b delivery ( Fig. 3a and supplementary  fig. 9). Actually neither figure shows delivery efficiency. 8. line 210 … and were intravenously injected with 20 nmol Ppm1b protein twice Material and Methods says that 5 nmoles (line 451). Which dose was actually used? These authors highlight the reasons protein delivery to cells cannot easily be accomplished, even by CPPs. Endosomal entrapment (poor release from endosomes) and loss of CPP delivery activity in serum. They have developed a hybrid approach using a CPP, pH sensitive membrane active peptide combined with endosome-specific protease sites and a leucine zipper for dimerization to create a protein delivery vehicle that appears to deliver proteins to cell cytosol in vitro and in vivo. This is a very interesting study, and there are multiple insights that will be of broad interest to researchers in protein delivery. However, there are some issues the authors will need to address to strengthen the paper before publication. Re: Thanks very much for your appreciation on the value of our work. We have carefully revised the manuscript according to the comments from you and the other two reviewers. Point-bypoint responses to the comments are listed below.

Efficient intracellular delivery of proteins by a multifunctional
1) The most significant issue of the paper is shown in Supplemental Figure 1. These data show very clearly that better delivery does not mean that individual cells get more protein delivered to them. All constructs tested, including the very poor ones, deliver a similar amount of cargo to individual cells. The better constructs simply deliver this amount of cargo to more cells. This fact is not addressed in the main paper, yet it holds important clues to something very fundamental about protein delivery that we do not understand. Many researchers have observed the same phenomenon. Delivery is stochastic, and cooperative at the cell level, not at the individual endosome level. Improving protein delivery to cells is not about improving the efficiency of release from individual endosomes. It is not about delivery of more protein to individual cells. With respect to cargo delivery, cells act cooperatively. Improving delivery is about delivering a bolus of protein to a greater fraction of cells. Yet, this entire paper (and most other papers on cargo delivery) presents the problem and discusses the results it as if the modifications carried out incrementally improve the amount of protein delivered to each cell at the level of individual endosomes. This is not correct. The fundamentally important observation in Supplemental Fig 1 must be a central part of the whole paper. Introduction, results, and discussion.
2) The fluorescence microscopy images shown for delivery of full length GFP seem not to show the same effect as described in point 1 above. Is this true? The authors should explain this. Re: We sincerely appreciate your valuable comments, because your comment 1-2 touch the same issue, thus we sought to address them together as follow： I. We claimed that better construct increased the amount of protein delivered into each cell, however, as show in supplementary figure.1, it seemly only delivered this amount of cargo to more cells.
Firstly, the FACS analysis in original Fig.1e demonstrated that our modification, such as introduction of two cleavage sites (TINNe-GFP1-10-NLS), increased mean fluorescence intensity (MFI) of total treated cells, indicating that more protein has been delivered into cells. This observation was shown more clearly by the shifting of peak in the histograms, which suggest that more proteins were delivered into each cell (Please see Fig.R1a below). Thus, we reasoned that the enhancement of MFI analyzed by FACS could support the claim that better construct increased amount of protein delivered to each cell. In the previous research by Wimley Group (Kauffman et al. (2018) Nat Commun, 9: 2568, the level of MFI of total treated cells was to evaluate the amount of cargo delivered to each cell as well.
Nevertheless, as you correctly pointed out, the original supplemental figure.1 showed that enhanced MFI of cell population would be accompanied by increase of the percentage of GFP-positive cells. However, when in delivery of full length GFP, all the cells seemly exhibit GFP fluorescence with different intensity between various constructs (comment 2). We speculated that it was attributed to two potential drawbacks of the endonuclear split GFP assay in our original submission: lower fluorescence intensity of GFP after complementation and undetermined level of GFP11 in the nucleus. 1) Due to lower fluorescence intensity of GFP after complementation, only when enough escaping GFP1-10-NLS entered into nucleus, the level of GFP could reached its detection threshold, in other words, green fluorescence in the nucleus could be imaged by microscope or identified as the GFP positive by FACS. As shown in Fig.R1a and 1b, enhanced MFI within total cells mediated by higher concentration of TINNe-GFP1-10-NLS was accompanied by increase of percentage of GFP-positive cells. Similar phenomenon was observed in other study using Split-GFP assay as well (Lonn et al. (2016) Sci Rep, 6: 32301.). 2) On the other hand, due to absence of any indicator for expression of GFP11, even though original HEK-293T-GFP11 cell line was identified by FACS after transfection of GFP1-10-NLSexpressing plasmid previously, we speculated that the level of GFP11 was still lower (even not express) in the majority of HEK-293T-GFP11 cells. This problem probably caused that only a fraction of cells shows GFP fluorescence after treatment with GFP1-10-related proteins as well, although enough GFP1-10-NLS entered into nucleus.
To overcome the second drawback of original endonuclear split GFP assay in the original MS, we constructed a new HEK-293T GFP11 cell line, in which full length mRuby3 (red fluorescence) was introduced in the N terminal of Histone H3 and GFP11, thus the signals of Ruby could be used to determine the expression of GFP11, and detect the nucleus (Please see 1). Finally, we investigated the intensity of GFP in the updated assay, as shown in Fig.R1b below, novel HEK-293T-GFP11 shows higher MFI than that of original assay, when incubated with same concentration of TINNe-GFP1-10-NLS. In addition, FACS analysis (Please see Fig.R1f below) also show that almost all the novel HEK-293T-GFP11 cells exhibit green florescence in both concentration groups, although the MFI was lower when the cells were treated with 1 μM TINNe-GFP1-10-NLS. Collectively, we reason that our updated assay could avoid this confused observation as much as possible.
Given that results involved original Split-GFP assay caused confusion above, in this revision, we re-performed all relevant experiments based on our novel endonuclear Split-GFP assay (i.e., Fig.1b, 1c, 1d, 1f, 2d, 2i, 2j and 2k; Supplementary Fig.1, 3, 4, and 5). Consistent with the results in original MS, proteolytic removal of CPP-PMAPs in endosomes indeed increase the MFI of HEK-293T-GFP11 as well. However, differ from the original data, when introduction of INF7 or more modules (TI-, TIN-, TINe-, TINNe-), almost all the HEK-293T-GFP11 exhibit observed green fluorescence, and the TINNe-GFP1-10-treated cells showed highest MFI (Please see below or revised Fig.1d). Now we have replaced all the relevant figures in original MS with new set of data using novel assay, in particular revised supplementary Fig. 3 below (former supplementary Fig.1). The results in revised supplementary Fig. 3 also show that more proteins were delivered into cells with the addition of PMAP and proteolytic sites.
With respect to the discrepancy between the delivery of Split-GFP and full-length GFP, as explained above, it was mainly attributed to lower florescence intensity of GFP after complementation, while the intensity of full length GFP is strong enough to be observed in the almost all the treated cells by using microscope. We reason this concern could also be addressed after the introduction of updated Split-GFP assay (Please see below or revised Fig.1h).

II. We claimed that that modifications performed incrementally improve the amount of protein delivered to each cell at the level of individual endosomes. However, delivery is stochastic, and cooperative at the cell level, not at the individual endosome level.
With respect to this concern, please allow us to explain that the process of cargo delivery by CPPs could be divided into two steps generally: internalization (endosome formation) and subsequent endosomal escape (cytosolic delivery and nucleus localization). Hence, the final delivery efficacy depended on the efficiency of these two steps. For example, in the Split-GFP assay, the MFI of HEK-293T-GFP11 cells depends on the level of GFP1-10-NLS reaching into the nucleus, while level of GFP1-10-NLS relies on the level of internalized cargo and the level of the internalized after successful endosomal escape. The western analysis in Fig.1e has shown us the amount of total internalization of GFP1-10-NLS in the cell level is all about the same among different delivery system, hence the increased amount of GFP1-10-NLS in the nucleus could be attributed to enhanced delivery (endosomal escape) at the level of endosomal escape. Moreover, the similar MFI of the HEK-293T treated with various GFP-related proteins validated that the similar efficiency of internalization was presence among different delivery system, although addition of INF7 resulted in a slight increase of MFI (Please see above or revised Fig.1h). To avoid this confusion, we have integrated above results and corresponding description into the revised MS (Page 6, Line 134-140).
3) One serious problem with almost all protein delivery assays is a lack of absolute measures of delivery. For example, Figures 1c and 1d show mean fluorescence intensity as a ratio of intensity observed with various constructs, to that observed with a contract that delivers little to no GFP1-10. Relative MFI is the ratio of a measurement over a small number. The reader of this paper needs to know what MFI=10 means in terms of absolute amounts. One could lyse cells, and use absolute fluorescence of the lysate compared to standard solutions of known concentrations of GFP to determine the absolute amount of GFP1-10 delivered. But given the stochastic nature of delivery (Supplemental Figure 1) a microscopic technique might be better suited. This problem of quantitation is even worse for enzymes because their effect is greatly amplified. Re: Thanks for your valuable comments. We agree with you about the need for measurement of absolute amounts of cargo delivered into cells, in particular the Fig 1 and  2, in which the relative MFI was used as index of delivery efficiency. In other words, we should show readers how much GFP were internalized into every cell (i.e., inside and outside of endosomes) and how much GFP1-10-NLS escaped from the endosomes and localized into nucleus (i.e., endosomal escape) after treatment of various constructs.

I. The establishment of standard curve for full length GFP and GFP1-10-NLS
The standard curve for GFP was established by directly analyzing the fluorescence intensity of GFP protein of known concentration (Please see below or new supplementary Fig.10a). As for the establishment of the standard curve for GFP1-10-NLS, the progress curves for complementation of GFP1-10-NLS and GFP11 in vitro was investigated firstly. We found that the fluorescence intensity starts to increase at 20 min after complementation starts, and reached plateau period at 4 hours (Please see below or new upplementary Fig.10b). Accordingly, the standard curve of fluorescence intensity vs.GFP1-10-NLS concentration was identified at 4 hours after complementation starts(Please see below or new supplementary Fig.10c).

II. Quantitative determination of GFP inside the cell
The HEK-293T were treated with 1μM of GFP-related proteins in 1 mL serum-free DMEM for 3 h at 37 °C，followed by the medium containing proteins was aspirated, and the cells were washed 3 times with DMEM containing 10 U/mL heparin. As you suggested, cells were counted (1×10 5 cells), harvested and lysed, then the amount of full-length GFP in lysate was quantified by measuring its fluorescence and converting to intracellular protein concertation using corresponding standard curve. Intracellular protein concertation was calculated as described in previous study (Erazo-Oliveras et al. (2014) Nat Methods, 11: 861-867.). We demonstrated that approximately 57.6 μM of GFP was present inside HEK-293T cells treated with TINNeL-GFP. Notably, consistent with the increment of MFI (fold increase) in FACS analysis, the amount of internalized cargo by TINNeL system was about 4-fold higher than that of TINNe and TAT peptide (Please see below or new supplementary Fig.10d).

III. Quantitative determination of GFP1-10-NLS in the nucleus
Likewise, HEK-293T-GFP11 were treated with 1μM GFP1-10-NLS-related proteins in 1 mL serum-free DMEM for 3 h at 37 °C. At 12 h post incubation onset, the medium containing proteins was aspirated, and the cells were washed 3 times with DMEM containing 10 U/mL heparin. Cells were counted (1×10 5 cells), harvested and lysed, then the amount of GFP1-

With regard to relative MFI (fold increase), please allow us to emphasize that it is obtained by MFI of total cells (not a small number) treated with indicated proteins divided by that of total cells treated with corresponding cargo protein only.
This method is widely recognized and used in the CPP-based researches (Milech et al. (2015) Sci Rep, 5: 18329;Lonn, Kacsinta et al. (2016) Sci Rep, 6: 32301;Kauffman, Guha et al. (2018) Nat Commun, 9: 2568Evans et al. (2019) Nat Commun, 10: 5012.). Therefore, we reasoned that "Relative MFI" may be preferable index for readers who want to know how about our system/strategy in comparison with previous study. Nevertheless, as you suggested, in our revision, based on the absolute amounts of delivered-cargo and its corresponding relative MFI, it was identified the one unit of relative MFI of GFP-related proteins treated cells means 1.4 μM GFP inside the cells (Please see below or new supplementary Fig.11a), while in Split-GFP assay, one unit means about 0.4 μM GFP 1-10-NLS escaped from endosome and located into nucleus (Please see below or new supplementary Fig.11b). 4) The writing syntax and sentence structure is very poor throughout the manuscript. The revised paper will need significant editing and polishing of the English. To provide one example, the opening paragraph of the discussion has several sentences that are nonsensical at present. Re: Thanks for your helpful suggestion. The revised MS has now been proofread by a native English speaker doing active research in protein delivery, and the language has been edited and polished by Springer Nature Author Services before resubmission.
5) The paper contains a number of statements that are essentially unsupported speculation. I feel that they should be removed if they cannot be supported by some evidence from the literature or from these experiments. Such speculations include: Line 119-120 "The CPP dimer might engage with the cellular membrane through multivalent interactions, but with, serum albumin in a monovalent manner; thus, the competitiveness of the serum is alleviated." Lines 253-256 " Unexpectedly, endosomal escape efficiency of this dimer is quite low than observed at co-incubation approach of dfTAT30. The reason for this is unclear, it is possible that the effect of cargo molecules on the interaction between TAT with BMP (bis(monoacylglycero)phosphate)." Re: Thanks for your valuable suggestions. With respect to the first speculation, as suggested by Reviewer 2#, the interaction between T-GFP or TL-GFP with BSA was verified using HPSEC, and binding between BSA with both T-GFP and TL-GFP were observed. In addition, the delivery of T-GFP was dramatically decreased (61.3%) in the presence of 50 g/L BSA, while the delivery of TL-GFP was not significantly changed ( Fig.2b). Therefore, interaction between CPP and negatively charged molecules such as BSA could decrease the delivery efficiency of monomeric CPP, but not the dimer. Even though, we cannot identify whether both proteins associate only one molecule of albumin due to the limited resolution of HPSEC analysis. Therefore, revised this statement (Page 6, Line 142-156; Page 8, Line 172-176). Regarding the second statement, we agreed with reviewer's comment, and removed this speculation in the revised MS as suggested.
6) The animal experiments are very impressive and add a lot to this work. Again, we lack any quantitative measure of delivery. Is there a way to determine how much enzyme must be delivered to prevent the acute damage that drives the observed phenomenon? Re: Thanks for your helpful comments. We agree that quantitative measurement of delivered Ppm1b in various tissues (e.g., liver) would be helpful for us to better characterize the efficacy of our platform. Due to difficulty of determination (e.g., lack of reliable and highly sensitive ELISA assay) and complexity of metabolism in vivo, currently it was technically challenging for us to quantify the absolute amount of Ppm1b in vivo. Nevertheless, to solve your concerns as much as possible, we rather roughly compared relative delivery efficiency by two indirect approaches: tracking fluorescence dye-labelled proteins (e.g, eTAT-Ppm1b-Cy5), and immunohistochemistry.
For tracking fluorescence dye-labelled proteins, twenty-four hours after intravenous administration with equimolar dose of eTAT-Ppm1b-Cy5, TAT-Ppm1b-Cy5 or Ppm1b-Cy5, all mice were euthanized(n=3), their main organs were collected, and the fluorescence intensity was analyzed. eTAT-Ppm1b showed higher fluorescence intensity than T-Ppm1b in multiple organs with different extent, confirming its excellent delivery performance in vivo (Please see below or revised Fig.3f and 3g). Moreover, consistent with the results of ex vivo imaging, the relatively quantitative analysis by using immunohistochemistry (Please see below or revised Fig.3h), as you suggested (Comment 7), validates the high efficacy of eTAT-based delivery. These results and relevant description have been included in the revised MS (Page 11, Line 262-277). Representative microscope images (f) and florescence intensity (g) in the indicated organs of the BALB/c mice intravenously administered with Ppm1b-Cy5, TAT-Ppm1b-Cy5 and eTAT-Ppm1b-Cy5 (n=3).
Twenty-four hours post-administration, the mice were sacrificed, and fluorescence imaging of each organ was performed. h Five hours after injection with indicated constructs or PBS, the tissues were harvested and prepared as paraffin slides. The nuclei were stained with DAPI, and the delivered Ppm1b were detected using anti-6×His IgG as the primary antibody and Alexa 647-conjugated goat anti-mouse IgG as the secondary antibody. The fluorescence was captured via fluorescence microscopy. Yellow boxes in the images indicate magnified region. The data in g are expressed as means ± s.e.m: * P<0.05, ** P<0.01, *** P<0.001, **** P < 0.0001 and NS, no significant difference.
Likewise, due to lack of appropriate approach of absolute quantification, it is also a big technical challenge for us the identification of how much enzyme must be delivered to cells in vivo to prevent the acute damage. Even though, we sought to answer this question by identify how much eTAT-Ppm1b were required to achieve therapeutic effect. In this revision, we have investigated the impact of different dose of eTAT-Ppm1b on APAP induced-ALF in the mouse model. The administration of 5 nmol eTAT-Ppm1b protein twice in 2 h and 6 h post-APAP injection almost fully inhibited the drug-induced the elevation of the level of ALT and AST in the serum, thus the mouse was intravenously administrated with 2 to 6 nmol eTAT-Ppm1b twice in 2 h and 6 h post-APAP administration to identify the minimum dose required to achieve effect. When at the concertation below 5 nmol, the level of ALT and AST elevated obviously. We thus concluded that the effective inhibition on APAP induced-ALF was observed when twice dose of 5 nmol (~15 mg/kg) eTAT-Ppm1b adopted at least. This effective dose was safe for mouse as no significant change of ALT and AST in serum in our research was observed (Please see below or new supplementary Fig. 11). Moreover, this dose was moderate level for CPP-based drug via i.v. injection, as indicated in the previous study (i.e., the median lethal dose of the CPP-oligomer conjugates administered iv was between 210 and 250 mg/kg) (Cai et al. (2006)

7)
Can the authors use the Cy5 fluorescence to determine the amount of enzyme delivered to cells? Can fluorescence microscopy be done on tissue sections to directly demonstrate and quantify cargo delivery to cells in vivo. Re: We greatly appreciate your valuable suggestion. With regard to the determination of amount of enzyme delivered to L929 using Cy5 fluorescence, we reasoned that, as described in the section of absolute measurement of GFP, direct measurement of fluorescent-tagged proteins can't specifically discriminate between endosomal escape and endosomal entrapment. In other words, it cannot reflect the amount of cytosolic delivery. Nevertheless, inspired by this comment and Reviewer 3# 's comment (the last one), in this revision, to investigate the efficacy of eTAT system when in the delivery of Ppm1b, we compared the relative level of Ppm1b delivered by eTAT and other control system at two key time point by western blotting. The results of 30 min post treatment could indicate the level of total cellular internalization (cytosolic delivery+ endosomal entrapment), while that of 12 h post treatment would indirectly reflect the level of cytosolic delivery (not all the cytosolic delivery), due to the entrapped proteins in the endosomes would be degraded quickly in the lysosome. As shown in Revised Fig.3a or see below, at 30 min post treatment, the level of Ppm1b delivered by eTAT in L929 was highest and that of all other TAT-based constructs are about same except Ppm1b alone. When 12 h post incubation onset, the western blotting demonstrated that higher level of Ppm1b (cleaved form) was detected in the cells treated with eTAT-Ppm1b than TINNe-, and no Ppm1b protein could be detected in cells treated with other TAT-based constructs. The efficient cytosolic delivery of eTAT-Ppm1b at 12 h post incubation onset was attributed to enhanced cellular uptake and more efficient endosomal escape. In general, these data suggest the eTAT show the highest efficiency for Ppm1b delivery. Now, we have integrated above results and corresponding description into the revised MS. (Page 10, Line 237-241). With regard to in vivo delivery, we performed immunostaining on paraffin sections of caecum and liver (two organs in which Ppm1b-related proteins were mainly distributed), at 5 h following a single injection of eTAT-Ppm1b and other control constructs. Our results showed that significant red fluorescence signal was observed in the caecum and liver of eTAT-Ppm1b-treated mice and the intensity was stronger than that of T-Ppm1b. On higher magnification, Alexa-647 signal of eTAT-Ppm1b was bright and diffused in the cytosol of tissue cells, confirming in vivo intracellular protein delivery (Please see below or revised Fig. 3h). Collectively, our results demonstrate that eTAT can deliver functional proteins Ppm1b more efficiently to cells in vivo. We have integrated above results into the revised manuscript, and greatly appreciate the reviewer's constructive suggestion (Page 12, Line 269-276).

Revised Fig. 3 eTAT-Ppm1b suppresses TNF-induced necroptosis in vitro and in vivo. h Five hours
after injection with indicated constructs or PBS, the main tissues were harvested and prepared as paraffin slides. The nuclei were stained with DAPI, and the delivered Ppm1b were detected using anti-6×His IgG as the primary antibody, and Alexa 647-conjugated goat anti-mouse IgG as the secondary antibody. The fluorescence was captured via fluorescence microscopy. Yellow boxes in the images indicate magnified region.

Yu et al. report on a novel CPP(TAT)-based intracellular protein delivery system (eTAT)
consisting of a CPP, PMAP, endosome-specific protease sites and a leucine zipper, showing enhanced cytosolic protein, phosphatase 1B (Ppm1b), delivery efficiency and serum tolerance in vitro and in vivo without toxicity, suppressing the TNF-α-induced systemic inflammatory response and acetaminophen-induced acute liver failure in the mouse model.
They provide a strategy to facilitate the development of CPP-based delivery platform on proteins for intracellular targets. The work is well performed and timely.
However, my concern is that it is difficult to see clear advantage of this method as compared to previously published protein delivery systems, e.g. TAT peptide and multiple others. I suggest to carry out additional studies in order to confirm such advantage. Re: We appreciated your positive summary and comments of our study. We are sorry for not clearly describing the advantages in our initial submission, this point touches one the same elements as raised by Reviewer #3 (point 1). Now we conclude the advantages of our delivery strategy and novelty of our study (Please see Response to the first comment of Reviewer #3). Briefly, one major finding in our study is providing a multi-modular CPPbased strategy for protein delivery to overcome the persistent drawbacks in the CPPsbased application. Accordingly, we choose the most widely used CPP, the translocation peptide derived from the HIV transactivator of transcription (TAT), as the prototypical CPP (T-cargo); thereby TAT-cargo is one of critical control delivery systems.
As reviewers suggested, to robustly confirm and clearly provide the advantages of our method, including enhanced delivery efficacy and serum tolerance in vivo, as compared to TAT-based delivery system or it containing other modules (e.g., INF7), we did additional experiments as listed below. We hope these additional data supplemented in the revision can enable our MS to win your satisfaction.

I. Refer to Revised Fig. 2 (or see below)
The data suggest that in compared with TAT-cargo, the dimerization of CPP-fused cargo mediated by leucine-zipper exhibits enhanced serum tolerance, mainly achieved by weakening the competitiveness of serum protein albumin, indicating that oligomerization of CPP-fused cargo was critical for its serum tolerance.

II Refer to revised Fig.3 (or see below)
The WB analysis demonstrated that the eTAT showed the highest level of Ppm1b delivery in L929 cell line, in compared with T-, TI-as well as TINNe-based system, suggesting the stronger delivery performance of eTAT system in vitro and for which all of four functional modules were responsible.

Revised Fig. 3 eTAT-Ppm1b suppresses TNF-induced necroptosis in vitro and in vivo. a
Immunoblot analysis of level of Ppm1b in L929 cells treated with Ppm1b-related proteins (1 μM) at indicated time point.

III. Refer to Revised Fig. 3 (or see below)
We demonstrated that eTAT-Ppm1b exhibited higher level of distribution in the multiple tissues, such as caecum, liver, of the BALB/C mouse based on the approach of the ex vivo imaging and immunohistochemistry, suggesting eTAT system is more potent than TAT in protein delivery in vivo.

Revised Fig. 3 eTAT-Ppm1b suppresses TNF-induced necroptosis in vitro and in vivo. f, g
Distribution of intravenously administered Cy5 labelled-Ppm1b-related proteins in the female BALB/c mice.
Twenty-four hours post-administration, the mice were sacrificed, and fluorescence imaging of each organ was performed. h Five hours after injection with indicated constructs or PBS, the tissues were harvested and prepared as paraffin slides. The nuclei were stained with DAPI, and the delivered Ppm1b were detected using anti-6×His IgG as the primary antibody and Alexa 647-conjugated goat anti-mouse IgG as the secondary antibody. The fluorescence was captured via fluorescence microscopy. Yellow boxes in the images indicate magnified region. The data in g are expressed as means ± s.e.m. * P<0.05, ** P<0.01, *** P<0.001, **** P < 0.0001, and NS, no significant difference.

Reviewer #3 (Remarks to the Author):
Authors have designed and refined Tat-peptide-based vector for delivering into cells protein cargoes covalently coupled to peptide. First, the widely used CPP was supplemented with PMAP sequence, and two protease cleaving sites for facilitating release of cargo proteins from endosomal entrapment. Later the dimerisation-inducing motif was included in peptide vector to increase the uptake by cells and reduce sensitivity towards serum albumin that is known to inhibit Tat-mediated delivery into cells. The efficiency of elaborated eTAT vector was verified by delivering GFP and its split version into model cell-lines. Finally, eTAT was used for delivery of Pmp1b to suppress TNF-induced necroptosis in vitro and in vivo. Moreover, eTAT-Pmp1b also increased survival of mice in acetaminophen-induced acute liver failure model. Re: We deeply appreciate your careful reading of our MS and the constructive comments provided. We have modified the MS accordingly. Major comments: 1. Authors claim that the used strategies for improving Tat-peptide mediated delivery are novel. However, higher delivery efficacy of multivalent CPPs compared to monovalent peptide has been earlier demonstrated by several laboratories e.g. of Futaki and Gariepy. Their works have to be referred to in introduction. The idea of using cleavage by protease for activation of CPP construct has been repeatedly exploited in the field of CPPs, and was pioneered by R. Tsien group, who perhaps also deserves mentioning. Re: Thanks for your valuable comment. As you correctly point out, the multivalent CPPs (MCPPs) and the strategy of proteolytic cleavage activated-delivery were reported in previous publications. As suggested, the relevant references have been cited in the revised MS. However, there is still significant novelty in the current study in comparison to related publications as described below. To solve your concerns, we have added clarifying statements in the main text of our revised MS to better describe our innovations with respect to what's been done previously. I. First of all，the most significant novelty of this paper relies on the novel multimodular CPP based-strategy for protein delivery. The four functional modules, constituted to one chimeric peptide delivery system, were all critical for the excellent delivery efficacy in vivo and in vitro. The sentences relevant to this novelty have been added in the introduction section of revised MS (Page 4, Line 71-78). II. Novel utilization of proteolytic cleavage-mediated activation. As you mentioned, the strategy of proteolytic cleavage activated-delivery has been widely utilized in the field of CPPs, after being pioneered by the group of Roger Y. Tsien for tumor imaging before (Jiang et al. (2004) Proc Natl Acad Sci U S A, 101: 17867-17872; Aguilera et al. (2009) Integr Biol (Camb), 1: 371-381.). For instance, proteolytic cleavage mediated by extracellular proteases (i.e., matrix metalloproteinases, MMP) specifically restores the penetrating activity of CPP moiety in the vicinity of tumor cells. However, these researches mainly focused on improvement of specificity in CPP-based delivery (He et al. (2016) J Control Release, 240: 67-76.). The strategy of proteolytic cleavage-based activation in our study, directed toward other intracellular enzymes, endosome-localized proteases (e.g., CTSL, furin), and was utilized to facilitate the endosomal escape. Due to the different intentions, the study of R. Tsien group was not mentioned in the original submission, now the relevant references have been added to the discussion section of revised MS as suggested (Page 14, Line 332-339). III. Novel approach for multivalent CPP-fused proteins formation. Many previous studies reported various strategies to prepare MCPP ) Biochim Biophys Acta, 1758, such as branched peptides system (Futaki et al. (2002) Biochemistry, 41: 7925-7930.) and attaching a protein oligomerization domain (e.g., p53tet) (Kawamura et al. (2006) Biochemistry, 45: 1116-1127 to CPPs. In the present study, we prepared multivalent CPP-fused protein by using leucine zippers motif for the first time. After purification of the TAT-GFP containing leucine zippers peptides (TL-GFP), it could selfassemble into a dimeric CPP-cargo. Similar dimer formation has been observed when cargo was GFP1-10-NLS or Ppm1b as well, indicating that leucine zipper mediateddimerization would be a convenient strategy to generate multivalent CPPs. The relevant researches (including that of Futaki and Gariepy) have been referred in the introduction section of revised MS as you suggested (Page 3, Line 66-70).

IV. Novel solution to serum intolerance:
In the previous study, MCPP was usually employed to increase the cellular uptake of CPPs (Kalafatovic and Giralt (2017) Molecules, 22: .), limited researches focus on its impact on serum tolerance. In the current study, we provided a proof-of-concept that the oligomerization of CPP-fused cargo was critical for its serum tolerance, which is probably achieved by weakening the competitiveness of serum protein albumin. The sentences relevant to this novelty have been included in the discussion section of revised manuscript (Page 15, Line 359-364). 2. Fig. 1a, a scheme of recombinant cell-penetrating protein, does not reveal where the polyhistidine tag that is used for purification of protein, is placed in the sequence. Please add! All data in manuscript suggest that histidine tag is still present on all used protein constructs, and it is not removed. Histidine residues are known to serve as a proton sponge and facilitate escape from endosomes, was its impact considered? Re: Thanks for your valuable comment. Firstly, we apologies for the omission of symbol of polyhistidine tag (His-tag) in original submission, which has been added for all constructs now. Due to the presence of His-tag in all recombinant proteins, thus we have not considered its effect before. As you correctly indicated, histidine residues are known to serve as a proton sponge and facilitate escape from endosomes, thus poly-histidine sequences have been used as motifs to improve endosomal escape in trans-delivery (coincubation) of gene ) Biomaterials, 29: 2408-2414 or ribonucleoproteins (Del'Guidice et al. (2018) PLoS One, 13: e0195558.). However, up to we know, there was ever no study focusing on its impact on endosomal escape when fused to CPP-cargo proteins. Based on the previous literatures by Gariépy group (Mohammed et al. (2012) J Control Release, 164: 58-64.) and Doi group (Sudo et al. (2017) J Control Release, 255: 1-11.) aimed at the function of new PMAPs, the His-tag was present on all used constructs in their study as well, which may suggested its role in endosomal escape could be ignored. Nevertheless, to address your concern, we have now included new data to assess the effect of His-tag on the endosomal escape using our novel Split-GFP assays (Please see Reviewer 1# point 1). The TINNe-GFP1-10-NLS without six histidine residues (6H) was constructed and purified (Please see Fig. R2a and R2b below). Then, the HEK-293T-GFP11 cells were treated by 5 μM TINNe-GFP1-10-NLS without HIS-tag (TINNe-GFP1-10-NLS-6H (-)) and TINNe-GFP1-10-NLS (TINNe-GFP1-10-NLS-6H (+)). Our data show that there is no significant difference between MFI of HEK-293T-GFP11 treated by two proteins (Please see Fig. R2c below). Collectively, this experiments according your suggestion show that the presence of His-tag would not intervene the assessment of endosomal escape efficiency in current study. Results in c shown are means ± s.e.m: NS, no significant difference.
3. Fig 1h. The intensity of GFP signal is substantially higher for TI-GFP compared to T-GFP after incubation MA104 cells with the respective protein constructs. The efficiency of cationic CPPs is dependent on the charge of peptide (R and K in sequence). Addition of INF7 to TAT sequence reduces the charge of targeting sequence from 8 to 4, which typically strongly impairs internalisation of peptide/protein construct. Here the fluorescence microscopy shows opposite -protein carrying the transducing sequence with lower positive charge is taken up by cells more efficiently than one with higher charge. Please explain this discrepancy in discussion part. Re: We appreciate this valuable comment. In Fig.1h of original MS, the fusion of INF7 to TAT sequence, which reduces the net charge of delivery sequence, conversely resulted in the enhancement of intracellular uptake. Actually, similar phenomenon was observed in previous researches   help us explain why constructs carrying the transducing sequence with lower positive charge is taken up by cells more efficiently than that of higher charge: firstly, positively charged residues R within CPPs indeed play a critical role in the uptake process, yet it has been demonstrated that guanidino moieties of R in delivering peptide played a greater role in facilitating cellular uptake than either the charge or the backbone structure (Mitchell et al. (2000) J Pept Res, 56: 318-325.); secondly, other factors, such as physical nature of fusedcargo also contribute to the extent of internalization. For example, two transducing sequence with different positive charge, when fused to cargo of small molecule TAMRA or GFP11 peptide, they exhibit reverse rank order of delivery efficiency (Kauffman, Guha et al. (2018) Nat Commun, 9: 2568.
Additionally, inspired by this comment, in order to better characterize the internalization efficiency various GFP-related recombinant proteins in the HEK-293T, the additional FACS analysis was included in the revised MS. Consistent with the observation of imaging, FACS analysis showed that the addition of INF7 enhanced, but slightly, the internalization efficiency of T-GFP (Please see below or revised Fig.1h). 4. Line 123 … As analyzed by SDS-PAGE, T-GFP and TL-GFP are monomers and dimers respectively (Fig. 2b). It is highly improbable that in SDS-PAGE the latter protein construct is in the form of dimer only. The Supplementary Figures 5, 6 and 8 show quite opposite. Re: Thanks for your comment. As we know, in the SDS-PAGE analysis, SDS would influence on the dimerization of leucine zipper-fused protein in different extent, depending on the physical nature of fused-cargo proteins and other motifs. We speculated that, when leucine zipper was fused to T-GFP, the formation of dimer is still enough stable in SDS-PAGE (without boiling), thus TL-GFP could be in the form of dimer only (or majorly). However, when fused to other proteins (e.g., T-GFP1-10), leucine zipper induced-dimer formation may be easier to be affected by SDS, thereby, as shown in SDS-PAGE of original Supplementary figures. 5, 6 and 8, only a fraction of leucine zipper-fused protein keeping leucine zipper motif induced-homogeneous dimerization. This is why dimer formation was investigated by using SDS-PAGE and HPSEC in our study. Prompted by your comments, we added the HPSEC analysis of TL-GFP and TL-GFP1-10 in this revision, which was missing in original submission (Please see below or revised Fig.2c, revised supplementary  Fig. 7b). 5. Line 191 … Twenty-four hours after administration, eTAT-Ppm1b was mostly distributed in the caecum … This is rather surprising observation. Has that been shown for other CPPs or CPPprotein constructs earlier? Please comment on this in discussion. Re: We appreciate your excellent comment. It is interesting observation for us, in this revision, we sought to further investigate whether or not eTAT peptide induce caecal distribution of eTAT-Ppm1b.

Revised Fig. 2 Dimerization of CPP-fused proteins enhances endocytosis and serum tolerance. c
Initially, we measured florescence intensity in multiple organs of mouse injected with Cy5labelled constructs, it was suggested the Ppm1b-Cy5, T-Ppm1-Cy5 and eTAT-Ppm1-Cy5 all mostly distributed in the caecum (Please see Fig.R3a and 3b below), which was never observed in previous CPP-based researches up to we know. To clarify whether eTAT or Ppm1b proteins would target to caecum, and resulting in caecum distribution, we delivered Cy5-labelled eTAT-GFP to BALB/C mice by intravenous instillation. We demonstrated that there is extremely lower Cy5 signal observed in the caecum of eTAT-GFP treated mice, while the similar level of florescence intensity was detected in the corresponding organs of eTAT-Ppm1b-treated mouse. (Please see Fig.R3a and 3b below).
Moreover, we further identify these observations using immunohistochemistry. The female BALB/C mice was intravenously administrated with Ppm1b, T-Ppm1b, eTAT-Ppm1b and eTAT-GFP. At 5 hours after treatment, the tissues were harvested and prepared as paraffin section. The delivered Ppm1b were detected using anti-6×his IgG as the primary antibody and Alexa 647-conjugated goat anti-mouse IgG as secondary antibody. Consistent with what was observed in ex vivo imaging. Our results showed that significant red fluorescence signal was observed in the various tissues, especially in the caecum, of eTAT-Ppm1b-treated mice and the intensity was stronger than that of T-Ppm1b (Please see below Fig.R4c). Moreover, there is extremely lower Alexa 647 signal was detected in the caecum of eTAT-GFP treated mice (Please see below Fig.R4c). These results suggest that the Ppm1b，not eTAT or any modules within it, induce the extraordinary caecum distribution of eTAT-cargo. The reason behind this phenomenon is clearly an area for our future research. We have integrated above results into the revised MS followed the suggestion of the reviewer (Page 12, Line 277-282), added this information in the discussion section and clearly stated that further experiments are needed to verify the reasons (Page 16, Line 387-391). Five hours after injection with indicated constructs or PBS, the tissues were harvested and prepared as paraffin slides. The nuclei were stained with DAPI, and the delivered cargo proteins were detected using anti-6×His IgG as the primary antibody and Alexa 647-conjugated goat anti-mouse IgG as the secondary antibody. The fluorescence was captured via fluorescence microscopy. Yellow boxes in the images indicate magnified region. Results in c shown are means ± s.e.m: *** P<0.001, **** P < 0.0001, and NS, no significant difference.
6. Line 234 … the endocytosis of the cargo was mediated by the interaction between TAT and the cell membrane (Fig. 5a) Does TAT peptide/motif interact directly with membrane? It has been repeatedly shown that proteins are involved in interaction and penetration of this CPP across membranes Re: We apologize for these confusing sentences. The endocytosis of CPPs with cells is initiated by interaction with membrane components on the cell surface, including glycosaminoglycans (GAGs), protein receptors or phospholipids, not directly with membrane. To avoid this confusion, we have corrected these sentences into" the endocytosis of the cargo was mediated by the interaction between TAT and membrane components on the cell surface (e.g., glycosaminoglycans) " in the revised MS (Page 14, Line 322-323).
7. Discussion contains multiple factual errors and difficult-to-understand sequences. It requires revision with focusing on obtained results and leaving out ample putative developments of presented protein delivery vector. Re: We appreciated you for this suggestion. The discussion has been rephrased to better summarize our major findings, as well explain the novelty and limitations of our method. 8. In multiple places the claims of authors are presented without the respective reference/citation (e.g. line 44-45, 50 etc.). Re: We have checked all the references throughout the manuscript and greatly appreciate your valuable comment. 9. The manuscript contains many typos (e.g. line 33), citation errors (e.g. line 59, line 118 [18]) and sentences that are difficult to read (e.g. lines 24-28, 37-40, 250-254) or are left unfinished (e.g. lines 255-256). Re: We greatly thank for your efforts paid on reviewing our MS. We have carefully checked the typos and grammar errors throughout the manuscript. In addition, the revised MS has now been proofread by a native English speaker doing active research in protein delivery, and the language has been edited by Springer Nature Author Services before resubmission. Specific comments: 1. Line 52-53 … After endocytosis mediated by CPP, the majority (approximately 90%) of the cargos are entrapped inside endosomes. Does it mean that 10% liberates and is active? Please provide reference for 90%. In general, one or two logs lower escape/activity is considered realistic. Re: Thanks for your valuable comment. As you correctly pointed out, one or two logs lower escape/activity is realistic. In the revised MS, we amended the description to "the majority (e.g., approximately 99 % when in the delivery of Cre recombinase) of the cargos are entrapped inside endosomes" based on the review paper written by Dowdy group (Lonn and Dowdy (2015) Expert Opin Drug Deliv, 12: 1627-1636, and added this reference in the revised MS as suggested (Page 3, Line 55). 2. Line 70-72 … Therefore, we believe the eTAT system or its improved version composed of multifunctional chimeric peptide has potential to become a major enabling strategy for protein delivery in biological research and therapeutic application. Exaggeration, considering how large quantities of protein construct (per kg) were required to achieve effect in mouse model. Re: Thanks for your valuable suggestion, this point touches one the similar elements as raised by Reviewer 1 #. We sought to answer this question by using the APAP induced-ALF mouse model. Actually, in our preliminary study on TNF-α induced SIRS model (prevention model), we have demonstrated that the minimal dose of eTAT-Ppm1b achieving the 100 % survival ratio was 5 nmol (Data was not shown in original MS, please see Fig.R4 below) followed by the comparison of different Ppm1b-related constructs. Accordingly, the dose of 5 nmol (twice) was employed in the APAP induced-ALF model (therapy model).
In the revised MS, to identify minimal does of eTAT-Ppm1b required to achieve therapeutic effect, the mouse was intravenously administrated with 2 to 6 nmol eTAT-Ppm1b twice in 2 h and 6 h post-APAP administration. When at the concertation below 5 nmol, the level of ALT and AST elevated obviously. We thus concluded that the effective inhibition on APAP induced-ALF was observed when twice dose of 5 nmol (~15 mg/kg) eTAT-Ppm1b adopted at least. This effective dose was safe for mouse as no significant change of ALT and AST in serum in our research was observed (Please see below or new supplementary Fig. 17). Moreover, this dose was moderate level for CPP-based drug via i.v. injection, as indicated in the previous study (i.e., the median lethal dose of the CPPoligomer conjugates administered iv was between 210 and 250 mg/kg) (Cai, Xu et al. (2006) Eur J Pharm Sci, 27: 311-319; Jarver, Mager et al. (2010) Trends Pharmacol Sci, 31: 528-535.)(Please see below or new supplementary Fig. 16). These results and relevant description have been included in the revised MS (Page 13, Line 299-304) Figure R4. Identification the minimum dose of eTAT-Ppm1b required to achieve 100 % survival ratio in female BALB/C. Wild-type female BALB/c mice were intravenously administrated with 3 to 6 nmole eTAT-Ppm1b followed by TNF challenge. Mouse survival was monitored every hour for 40 h, with the results presented in a Kaplan-Meier plot, and a log-rank test was performed; n=8 mice for each group. * P<0.05, **** P < 0.0001 and NS, no significant difference. figure. 16 Identification the minimum dose of eTAT-Ppm1b required to achieve therapeutic effect in female BALB/C. a Wild-type female BALB/c mice were intravenously administrated with 2 to 6 nmol eTAT-Ppm1b twice in 2 h and 6 h post-APAP administration. b-c Serum was collected at 12 h post APAP administration, then activities of (b) ALT and (c) AST were determined by routine clinical assays using commercial kits. Results shown are means ± s.e.m: n = 7 mice for each group. **** P < 0.0001 and NS, no significant difference. Figure 1e type of actin? The level of actin varies substantially between lines. Please specify (in Materials and Methods) how the amount of protein that was applied to gel was equalised. Re: We apologies for this omission. β-actin was used as loading control, and we corrected the label "Anti-actin " to "Anti-β actin" in Fig.1e in the revised MS.

In
To equalize the amount of protein which was applied to gel, firstly, same number of (5 ×10 5 cells per well in 12-well plates) were seeded in cell culture plates. Next, after treatment with the conditions specified in the figure legends, all cells in the plate were detached, resuspended in PBS, and the cell numbers were determined and adjusted to 1×10 5 cells per 100 μL lysis buffer for WB analysis, and equal volume was loaded to each lane. Now we have specified this process in Materials and Methods as reviewer suggested (Page 21, Line 490-491, 492-495).
In the Fig.1e, cells were treated with different GFP1-10-related proteins at the same time, then collected at the indicated time point. Thus, the variation of the β-actin level between lines, especially 3 h/6 h with other time point, may be attributed to that anti-β-actin antibody was recovered from experiments in 0.5 h and 1 h. Nonetheless, due to similar level of βactin showed in different lanes, so we reason that our conclusion could be supported by current data. 4. Figure 1f. Fluorescence of GFP in split assay peaks at 9 h time-point and starts to decrease from there, which occurs surprisingly quickly. Please comment this in discussion. Re: Thanks for your valuable comment. As shown in Fig.1e, the western blotting analysis suggested that a fraction of complete GFP constituted of GFP1-10 and GFP11 have degraded between 9 h and 12 h post-incubation onset. Accordingly, fluorescence intensity of GFP decrease quickly from 9 h post-incubation onset. To further validate this observation, we have repeated this experiment using novel Split-GFP assay, and have updated the results in Fig. 1e. The new results suggest a similar pattern of kinetics of green fluorescence (Please see below or revised Fig.1f). 5. Lines 118-119 … homodimerization capacity was employed to mediate the dimerization of the CPP-fused proteins. This approach has been used with CPPs earlier. Please add reference. Re: Thanks for your helpful suggestion. The relevant references have been cited in the revised MS. 6. Lines 119-121 … The CPP dimer might engage with the cellular membrane through multivalent interactions, but with, serum albumin in a monovalent manner; thus, the competitiveness of the serum is alleviated. What gives ground for this assumption? Has it been shown experimentally? For verifying this assumption, the authors are advised to analyse the elution of dimeric protein construct (after incubation with serum or not) from SEC column. Comparison of that with elution profiles of monomeric from should reveal, whether both proteins associate only one molecule of albumin. Re: This is an excellent advice. The suggested experiment has now been performed in the revised MS. It has been proposed previously that CPP could binds to negatively charged proteins in the serum, such as albumin (Kosuge et al. (2008) Bioconjug Chem, 19: 656-664.) (accounted for 35-50g/L in the serum), and which would suppress the CPPsmediated endocytosis (Mueller et al. (2008) Bioconjug Chem, 19: 2363-2374. But it was just speculation and there was still no direct evidence that CPPs could bind to BSA and thus suppress the CPPs mediated endocytosis. As you suggested, we have now verified the binding between TAT motif and BSA (bovine serum albumin), which is the richest components in serum, by using HPSEC (Please see below or new supplementary Fig. 6a). Initially, 25 μM GFP or T-GFP were incubated with 75 μM BSA individually (molar ratio=1:3), the HPSEC analysis demonstrated that BSA only can bound to TAT motif of T-GFP (Please see below or new supplementary Fig. 6b and 6c). On the other hand, FACS analysis in the HEK-293T cells showed that the MFI of 5 μM T-GFP-treated cells in the presence of BSA (50 g/L) decreased to 38.7% of its counterpart in the serum-free DMEM (Please see below or revised Fig. 2a). Consistent with previous speculations, our data show that the interaction of albumin with TAT contributes to the low serum tolerance of TAT-mediated delivery. As expected, interaction between TL-GFP and BSA was observed (Please see below or new supplementary Fig. 6d) as well, however, MFI of TL-GFP-treated cells in the presence of BSA remained 83.1% of that in the free DMEM (Please see below or revised Fig. 2f), suggesting that dimerization of TAT could alleviate the binding of BSAinduced inhibitory effect on the endocytosis. Even though, due to the restricted resolution of SEC analysis in this research, we cannot identify whether both proteins associate only one molecule of albumin. Therefore, available data are not going to provide a definitive answer to the question if CPP interacts with serum albumin in a monovalent manner, further study needs to be performed to verify this assumption. Accordingly, we now deleted this speculation as suggested and rephrased this statement (Page 6, Line 142-156; Page 8, Line 172-176). 7. Line 170 … eTAT showed the highest efficiency for Ppm1b delivery (Fig. 3a and  supplementary fig. 9). Actually neither figure shows delivery efficiency. Re: Thanks for your valuable suggestion. In the original submission, we speculated the proportion of survival L929 post treatment with the mouse TNF-α and zVAD (TNZ), has positive correlation with the level of Ppm1b in the cytosol (Chen et al. (2015) Nat Cell Biol, 17: 434-444.). Therefore, we only detected PI uptake efficiency of L929 after treatment to evaluate the delivery efficiency. However, as you correctively pointed out, it was not a direct evidence of higher delivery efficiency. To solve this issue, in this revision, we have evaluated the level of Ppm1b in the L929 treated with eTAT-Ppm1b and other constructs directly by western blotting at two key time point by western blotting. As shown in new Fig.3a below, the results of 30 min post treatment could indicate the level of total cellular internalization (cytosolic delivery + endosomal entrapment), while that of 12 h post treatment would indirectly reflect the level of cytosolic delivery (not all the cytosolic delivery), due to the entrapped proteins in the endosomes would be degraded quickly in the lysosome. As shown in revised Fig.3a or see below, at 30 min post treatment, the level of Ppm1b delivered by eTAT in L929 was highest and that of all other TAT-based constructs are about same except Ppm1b alone. When 12 h post incubation onset, the western blotting demonstrated that higher level of Ppm1b (cleaved form) was detected in the cells treated with eTAT-Ppm1b than TINNe-, and no Ppm1b protein could be detected in cells treated with other TAT-based constructs. The efficient cytosolic delivery of eTAT-Ppm1b at 12 h post incubation onset, was attributed to enhanced cellular uptake and more efficient endosomal escape. In general, these data suggest the eTAT show the highest efficiency for Ppm1b delivery. Now, we have integrated above results and corresponding description into the revised MS (Page 10, Line 237-241).
by  showing conformational changes of the conjugated sepeptide which might promote cell membrane interaction. I have a doubt on this explanation. Tat is able to internalize big proteins as well as small peptides. I would rather believe that the INF7 peptide acts as spacer or linker making Tat more accessible for the interaction with the cell surface GAGs. If the authors believe on the conformational change of Tat versus Tat-INF7 -both peptides could be analyzed by circular dichroism in order to see changes and to confirm their hypothesis.
Re: Thank you for these helpful comments. Firstly, we completely agreed with you that our observation could be attributed to that INF7 peptide acts as spacer or linker making TAT more accessible for the interaction with the cell surface GAGs, which would be one of the possible mechanisms behind this observation. Secondly, with regard to our explanation for the conformational change of TAT in previous revision, due to their data  Pharmaceuticals (Basel), 2: 49-65.) was obtained by investigating the difference of cellular uptake of carboxyfluorescein (CF)-labelled TAT or TAT-PMAP, accordingly, we indicated that it was a one of probable mechanism behind this observation although we did not know whether this conformational change occurs when fused to cargo proteins. With regard to your suggested experiments that both peptides (TAT, TAT-INF7) could be analyzed by circular dichroism to see changes and to confirm this hypothesis. We have discussed with the experts doing active research in protein structure, and they suggested that the molecular size of TAT (13 aa) was too small in comparison with its fused-cargo proteins (ie,GFP, 238 aa ), and it was difficult to accurately identify whether the conformational change of TAT appear in the TAT-INF7-cargo proteins by using this approach. Even though, based on these comments, in this revision, your suggestion, and our original explanation, were clearly described as two potential mechanisms behind this observation, and we believed this could be addressed in the future studies. The following sentences were included in revised manuscript (Line 354-359): "The increases in cytosolic uptake by fusion of PMAP (HA2) to CPP has been also observed by Wölfl group and they proposed that the enhancement was due to conformational changes (Neundorf, Rennert et al. (2009) Pharmaceuticals (Basel), 2: 49-65.), which may enhance the membrane interaction or endosome disruption. Besides, the INF7 peptide could also act as a spacer or linker, which may make TAT more accessible for the interaction with the cell surface components (ie, GAGs), and thus increase the intracellular uptake." Question 4 of reviewer #3: I agree completely with the response of the authors. Indeed, in particular cases protein dimerization could be observed in SDS-PAGE even under denaturating conditions. Re: We appreciated you for these positive comments.
Question 5 of reviewer #3: eTAT-Ppm1b distribution in the caecum -as required by reviewer #3, the authors have commented this fact in the result and discussion sections.
Re: Thank you for these comments.